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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-06-09 to 2015-07-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
1993
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
TA 98 (his D 3052, uvrB, rfa + R-factor)
TA 100 (his G 46, uvrB, rfa + R-factor)
TA 1535 (his G 46, uvrB, rfa)
TA 1537 (his C 3076, uvrB, rfa)
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : Male Wistar, HSdCpb:Wu rats, aged 6-8 weeks, were given a single intraperitoneal injection of Aroclor 1254 (500 mg/kg body weight) dissolved in Miglyol 812 oil (Merck KGaA, Darmstadt, Germany). The animals received drinking water and a standard diet ad libitum. The body weight of the animals used was 179 g ± 6.76 g.
- method of preparation of S9 mix : according to Ames
- quality controls of S9: Every S9-batch is tested for its metabolic activity by the use of specific substrates, requiring different enzymes of the P450-isoenzyme family. The mutagenicity of 2-aminoanthracene, benzo[a]pyrene, and 3-methylcholanthrene is thus determined once for every S9-batch.
Test concentrations with justification for top dose:
with and without S9: 5.00, 15.8, 50.0, 158.0, 500.0, 1580.0, 5000.0 µg/plate
Vehicle / solvent:
- Vehicle/solvent used: DMSO

- Justification for choice of solvent/vehicle: well solubility of the test item; recommended by the guidelines
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
sodium azide
other: Daunomycin; 2-Aminoanthracene
Rationale for test conditions:
According to the guidelines
Evaluation criteria:
The assessment of test material-induced effects is dependent on the number of spontaneous revertants of each bacterial strain (solvent controls) and the increase in the number of revertants at the test material concentration which shows the highest number of colonies. The criteria provided in table 1 in "any other information on materials and methods", based upon the historical controls of the laboratory and statistical considerations, were established.

Interpretations:
A test material was to be defined as negative or non-mutagenic in this assay if
• the assay was to be considered valid, and
• "no" or "weak increases" occurred in the test series performed ("weak increases" randomly occur due to experimental variation)

For valid data, the test material was considered to be positive or mutagenic if:
• the assay was to be considered valid, and
• a dose dependent (over at least two test material concentrations) increase in the number of re-vertants was induced, the maximal effect was a "clear increase", and the effects were reproduced at similar concentration levels in the same lest system or
• "clear increases" occurred al least at one test material concentration, higher concentrations showed strong precipitation or cytotoxicity, and the effects were reproduced at the same concentration level in the same test system.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: no precipitation observed

Any other information on results incl. tables

Please refer to background material attached.

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study the test item is considered mutagenic in procaryotic cells.
Executive summary:

The investigations for the mutagenic potential of the test item were performed using Salmonella typhimurium tester strains TA 98, TA 100, TA 1535 and TA 1537, and Escherichia coli WP2 uvrA. The plate incorporation test with and without addition of liver S9 mix from Aroclor 1254-pretreated rats was used. Two independent experimental series were performed. In the two series with S9 mix, 10 % S9 in the S9 mix were used in the 1st and 30 % in the 2nd series, respectively.

All test item treatments in this study were performed using formulations prepared in anhydrous analytical grade dimethyl sulphoxide (DMSO). Treatments of all tester strains were performed in the absence and in the presence of S9 mix, using final concentrations of the test item between 5 and 5000 µg/plate, plus vehicle and positive controls. After test material exposure, no precipitation of the test material on the agar plates occurred and no toxicity to the bacteria was observed.

Daunomycin, sodium azide, 9-aminoacridine, and 4-nitroquinolin-N-oxide served as strain specific positive control test materials in the absence of S9 mix. 2-Aminoanthracene was used as positive control in the presence of the metabolic activation and thus for testing the activity of the S9 mix.

Vehicle and positive control treatments were included for all strains. The mean numbers of revertant colonies all fell within acceptable ranges for vehicle control treatments, and were clearly elevated by positive control treatments, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used.

Under the conditions of this assay, there were relevant increases in revertant numbers after test item exposure observed in TA 100 in the absence of S9 mix in both series.

Therefore, the test material is considered mutagenic under the described experimental conditions.